CN103512984A - Sample pretreatment method for detecting different clenbuterol residuals - Google Patents
Sample pretreatment method for detecting different clenbuterol residuals Download PDFInfo
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- CN103512984A CN103512984A CN201310496272.0A CN201310496272A CN103512984A CN 103512984 A CN103512984 A CN 103512984A CN 201310496272 A CN201310496272 A CN 201310496272A CN 103512984 A CN103512984 A CN 103512984A
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Abstract
The invention relates to a sample pretreatment method for detecting different clenbuterol residuals. In the method, no halogen preparation is used, liquid-liquid extraction processes after alkalization are reduced, and the operation process is simple and fast. The method adopts an enzymolysis, extraction and purification manner for sample pretreatment, particularly, a zymolytic sample is directly purified through an MCX solid phase extraction column after being extracted through acidified methyl alcohol, and detection is performed through liquid chromatography tandem mass spectrometry. Twenty four samples can be detected within 6 hours, so that the detecting time is saved by about half, and the detecting cost is also lowered greatly.
Description
Technical field:
The present invention relates to a kind of biological detecting method, be specifically related to the residual sample-pretreating method of a kind of detection variety classes " clenbuterol hydrochloride ".
Background technology:
" clenbuterol hydrochloride ", is being commonly called as of clenobuterol hydrochloride at first, now makes a general reference the illegal class material containing β-adrenergic receptor activator class with promotion growth of animal, raising lean meat percentage using of aquaculture.Comprise that many countries of China are all to β in food animal
2strict supervision has been implemented in the illegal use of-receptor stimulating agent, since two thousand two, China with regard to successively issue No. 176, No. 193, No. 235 bulletin, within 2010, issue again bulletin No. 1519, clearly provided against β such as using clenobuterol hydrochloride, salbutamol, salbutamol sulfate, Ractopamine, Dopamine hydrochloride, Cimaterol, bricalin, phenolethanolamine A, bambuterol, hydrochloric acid Zilpaterol, clorprenaline hydrochloride, Mabuterol, western Boot sieve, bromine Boot sieve, tartrate Afromoterol, formoterol fumarate
2-receptor stimulating agent and clonidine hydrochloride, anarexol etc. amount to 18 kinds of " clenbuterol hydrochloride " medicines.
In order to strengthen the supervision to " clenbuterol hydrochloride " medicine, scientific research personnel has carried out research widely for " clenbuterol hydrochloride " residual detection method in animal food both at home and abroad.At present, pay close attention to the more 3 kinds of medicines such as Clenbuterol, salbutamol, Ractopamine that mainly contain, the detection method of other drug is relatively lagged behind; And at home and abroad there is no report to detect the analytical approach of novel " clenbuterol hydrochloride " medicament residues such as clonidine, cyproheptadine, phenolethanolamine A, Clorprenaline simultaneously.
Though issued β a plurality of detection animal tissue from examination criteria Lai Kan， China
2the examination criteria of-receptor stimulating agent, as: " in animal derived food, the standard such as No. 1025 bulletin-18-2008 of the mensuration Liquid Chromatography-Tandem Mass Spectrometry Fa》, Ministry of Agriculture of multiple beta-receptor activator residual quantity " beta-receptor activator residue detection Liquid Chromatography-Tandem Mass Spectrometry in animal derived food ", also only plants β for more than 10 to GB-T22286-2008
2the detection of-receptor stimulating agent, and in sample pretreatment process, liquid-liquid extraction program is complicated, complex operation, takes time and effort, and staff's operative technique is had relatively high expectations, and the accuracy of testing result and degree of accuracy are had a direct impact; In addition, in process of the test, also used a large amount of halogen preparations (perchloric acid), as very easily environment and biology caused to larger impact to liquid waste processing is improper.
In modern detection of veterinary drugs in food, the large-scale instrument and equipments such as Liquid Chromatography-Tandem Mass Spectrometry instrument are increasingly advanced, sample pre-treatments is even more important as committed step, the preci-sion and accuracy of direct impact analysis result, select suitable pre-treating method, shorten the pre-treatment time of sample, therefore, good Sample Pretreatment Technique is Yi Ge important channel and the key link that guarantees to detect quality, improves detection efficiency.
There is the problems such as testing process is loaded down with trivial details, agents useful for same has greater environmental impacts, operative technique is had relatively high expectations in current analytical approach.Testing agency, when practical application, easily occurs that testing result is unstable, and preci-sion and accuracy is not high, easily causes the erroneous judgement to testing result, occurs undetected or false-positive phenomenon, thereby causes administrative service division to supervise ineffective to " clenbuterol hydrochloride ".
What is more important, still do not detect at present both at home and abroad the effective ways of these " clenbuterol hydrochloride " medicines that the Ministry of Agriculture bans use of simultaneously, thereby, supervision for the illegal use of novel " clenbuterol hydrochlorides " such as clonidine, cyproheptadine, phenolethanolamine A, Clorprenalines does not still have effective technological means, very easily causes " supervision vacuum ".
Summary of the invention
The β such as Clenbuterol
2-receptor stimulating agent and target tissue cell membrane β
2-receptors bind, and then there is its pharmacological action, be generally in vivo yoke and close state, especially the salicylic acid β such as salbutamol
2-receptor stimulating agent yoke composition and division in a proportion example is higher.Therefore,, before sample extraction, the determinand that conventionally need to yoke be closed to state by enzymolysis dissociates further to carry out liquid-liquid extraction.β
2-receptor stimulating agent is generally soluble in organic solvent under alkali condition, solution isopolarity solvent soluble in water under acid condition.Because many organic solvents easily cause larger pressure to environment, therefore, the present invention intends extracting with the methanol solution of acidifying, with an organic solvent few to try one's best, especially toxic solvent; Meanwhile, also take into account the dissolution characteristics of two kinds of medicines of clonidine and cyproheptadine in acidifying methyl alcohol, to reach the object of extraction and analysis simultaneously.
In order to set up the Liquid Chromatography-Tandem Mass Spectrometry method of forbidding " clenbuterol hydrochloride " medicine of using in food animal that detects all China Ministry of Agriculture regulation simultaneously, also overcome present analysis method testing process loaded down with trivial details simultaneously, halogen reagent used has greater environmental impacts, the operative technique problem such as have relatively high expectations, the invention provides and a kind ofly can detect animal blood simultaneously, muscle, liver, kidney, animal tissue's medium cokes such as lungs are fixed, cyproheptadine and phenolethanolamine A, Clorprenaline, Clenbuterol, salbutamol, Ractopamine, Cimaterol, Terbutaline, bambuterol, Zilpaterol, Mabuterol, western Boot sieve, bromine Boot sieve, Formoterol, special sieve of spraying, methyl Clenbuterol, salmeterol, plug Boot sieve, gram logical sequence third sieve, 19 kinds of β such as fenoterol
2-receptor stimulating agent amounts to the sample pretreatment process of 21 kinds of " clenbuterol hydrochloride " medicines.Operating process is simple, using amount of reagent is few, and reagent, the operation steps used follow environmentally friendly principle, to reduce as far as possible the destruction that environment is caused.
Due to β
2-receptor stimulating agent is easy and protein combination in animal body, and therefore, in retention analysis process, the main mode of " enzymolysis+extraction+purification " that adopts is carried out sample pre-treatments.After acidifying methyl alcohol extracts, directly utilize MCX solid-phase extraction column to purify in the sample through enzymolysis, Liquid Chromatography-Tandem Mass Spectrometry detects.
Specific embodiments:
Embodiment 1:
Accurately take the fluid samples such as 2g blood, body fluid or musculature in 50mL centrifuge tube, add ammonium acetate solution (0.2mol/L) 5mL of pH5.2, then add 20 μ L glucuronidase-sulfatase (sigma company) whirling motion 30s, put 37 ℃ and spend the night, after taking out next day
4the centrifugal 5min of 000rpm, gets supernatant and puts in another centrifuge tube; Residue adds 1% formic acid methanol solution 5mL, whirling motion 30s, and the centrifugal 5min of 4000rpm, merges supernatant, then adds 5mL1% formic acid methanol solution to extract once, merges supernatant, to be clean.
Embodiment 2:
Accurately take the tissue samples such as 2g liver, kidney, lungs in 50mL centrifuge tube, except adding 1% formic acid methanol solution 5mL, whirling motion 30s, outside the centrifugal 5min of 10000rpm, other steps are identical with embodiment 1.
Embodiment 3:
The solution of getting 5mL extraction adds the MCX Solid-Phase Extraction column purification through 3mL methyl alcohol, pure water activation, use successively 3mL pure water and methanol wash, MCX is purified to extraction column to be drained, with 3mL5% ammonification methanol solution wash-out, nitrogen dries up rear use 0.1% aqueous formic acid/acetonitrile (9:1, v/v) dissolve, after liquid chromatography-tandem mass spectrometry instrument detects (inner mark method ration), can obtain satisfied result.Concrete detected parameters is as follows:
1, the sensitivity of method: in blood, muscle, the detectability of 21 kinds of medicines can reach 0.1 μ g/kg, is quantitatively limited to 0.25 μ g/kg; The detectability of liver, kidney, 21 kinds of medicines of lungs tissue can reach 0.2 μ g/kg, is quantitatively limited to 0.5 μ g/kg, compares improve a lot or quite, can meet the requirement of " clenbuterol hydrochloride " supervision in animal food completely with existing detection method.
2, the degree of accuracy of method and accuracy: with the recovery of 21 kinds of medicines medicine under the interpolation concentration conditions of 0.5 μ g/kg, 1.0 μ g/kg and 2.0 μ g/kg in liver, the coefficient of variation (relative standard deviation, RSD) as shown in table 1.(the same)
3, the scope of detected parameters: current detection method can only detect clonidine and cyproheptadine or simple beta-receptor activator, and this method can detect above-mentioned different types of " clenbuterol hydrochloride " medicine simultaneously, the scope of the detection of expansion.
In table liver, 21 kinds of medicament residues detect the recovery and relative standard deviation
Above-mentioned experimental data explanation: the present invention, in sample pretreatment process, does not use halogen preparation, reduces the rear Liquid-liquid Extraction Processes of alkalization, operating process is simple, quick, in 6 hours, can complete the detection of 24 samples, save detection time near half, testing cost also reduces greatly.
Claims (5)
1. detect the residual sample-pretreating method of variety classes " clenbuterol hydrochloride ", it is characterized in that, described method adopts the mode of " enzymolysis+extraction+purification " to carry out sample pre-treatments.
2. method claimed in claim 1, is characterized in that the sample through enzymolysis, after acidifying methyl alcohol extracts, directly utilizing MCX solid-phase extraction column to purify, and Liquid Chromatography-Tandem Mass Spectrometry detects.
3. method claimed in claim 2, wherein said enzymolysis is: accurately take 2g sample in 50mL centrifuge tube, ammonium acetate solution (0.2mol/L) 5mL that adds pH5.2, add again 20 μ L glucuronidase-sulfatase (sigma company) whirling motion 30s, putting 37 ℃ spends the night, after taking out next day, the centrifugal 5min of 4000rpm, gets supernatant and puts in another centrifuge tube.
4. method claimed in claim 2, wherein said being extracted as: the residue after enzymolysis is added to 1% formic acid methanol solution 5mL, whirling motion 30s, the centrifugal 5min of 4000rpm, merges supernatant, then adds 5mL1% formic acid methanol solution to extract once, merge supernatant, to be clean.
5. method claimed in claim 2, wherein said purification is: the solution of getting 5mL extraction adds the MCX Solid-Phase Extraction column purification through 3mL methyl alcohol, pure water activation, use successively 3mL pure water and methanol wash, MCX is purified to extraction column to be drained, with 3mL5% ammonification methanol solution wash-out, nitrogen dries up rear use 0.1% aqueous formic acid/acetonitrile (9:1, v/v) and dissolves, and through liquid chromatography-tandem mass spectrometry instrument, detects (inner mark method ration).
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104807919A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Method for separating Clenbuterol from swine urine by adopting SLE (Supported Liquid Extraction) method |
| CN104807926A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Method for simultaneously separating ractopamine, clenbuterol and salbutamol from feed by adopting SLE method |
| CN104931600A (en) * | 2015-05-12 | 2015-09-23 | 广西壮族自治区梧州食品药品检验所 | Method for high-precisely measuring salbutamol in swine urine |
| CN105092762A (en) * | 2015-08-26 | 2015-11-25 | 中国农业科学院兰州畜牧与兽药研究所 | Method for measuring residual quantities of growth promoting agents such as zilpaterol, cimbuterol, clenproperol and bambuterol in beef |
| CN105277632A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Method for detecting cyproheptadine residual quantity in swine urine through high performance liquid-tandem mass spectrometry |
| CN105651869A (en) * | 2014-12-30 | 2016-06-08 | 青岛康大食品有限公司 | Method for extracting clenobuterol hydrochloride in pork |
| CN106769282A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | Left-handed 18 methylnorethindron detection method and kit in a kind of animal tissue |
| CN109612819A (en) * | 2018-11-28 | 2019-04-12 | 温州医科大学 | Based on pretreatment technology and its application associated with enzymatic hydrolysis auxiliary and solidification floating drop micro-extraction |
| CN110208394A (en) * | 2019-05-16 | 2019-09-06 | 上海康识食品科技有限公司 | The detection method of Ractopamine, salbutamol and Clenbuterol in a kind of poultry meat |
| CN112903873A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Free-state and conjugated-state salbutamol standard substance contained in swine urine freeze-dried powder after animal metabolism and preparation method thereof |
| CN112903874A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Free and conjugated clenbuterol standard substance contained in pig urine and preparation method thereof |
| CN112903875A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Ractopamine standard substance with conjugated state and free state coexisting in pig urine matrix as well as preparation method and application thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105277632A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Method for detecting cyproheptadine residual quantity in swine urine through high performance liquid-tandem mass spectrometry |
| CN105651869A (en) * | 2014-12-30 | 2016-06-08 | 青岛康大食品有限公司 | Method for extracting clenobuterol hydrochloride in pork |
| CN104807926B (en) * | 2015-05-12 | 2016-11-23 | 广西壮族自治区梧州食品药品检验所 | Use the method that SLE method concurrently separates the Ractopamine in feedstuff, clenbuterol, albuterol |
| CN104807926A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Method for simultaneously separating ractopamine, clenbuterol and salbutamol from feed by adopting SLE method |
| CN104931600A (en) * | 2015-05-12 | 2015-09-23 | 广西壮族自治区梧州食品药品检验所 | Method for high-precisely measuring salbutamol in swine urine |
| CN104807919A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Method for separating Clenbuterol from swine urine by adopting SLE (Supported Liquid Extraction) method |
| CN105092762A (en) * | 2015-08-26 | 2015-11-25 | 中国农业科学院兰州畜牧与兽药研究所 | Method for measuring residual quantities of growth promoting agents such as zilpaterol, cimbuterol, clenproperol and bambuterol in beef |
| CN106769282A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | Left-handed 18 methylnorethindron detection method and kit in a kind of animal tissue |
| CN109612819A (en) * | 2018-11-28 | 2019-04-12 | 温州医科大学 | Based on pretreatment technology and its application associated with enzymatic hydrolysis auxiliary and solidification floating drop micro-extraction |
| CN110208394A (en) * | 2019-05-16 | 2019-09-06 | 上海康识食品科技有限公司 | The detection method of Ractopamine, salbutamol and Clenbuterol in a kind of poultry meat |
| CN112903873A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Free-state and conjugated-state salbutamol standard substance contained in swine urine freeze-dried powder after animal metabolism and preparation method thereof |
| CN112903874A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Free and conjugated clenbuterol standard substance contained in pig urine and preparation method thereof |
| CN112903875A (en) * | 2021-01-25 | 2021-06-04 | 中国农业科学院农产品加工研究所 | Ractopamine standard substance with conjugated state and free state coexisting in pig urine matrix as well as preparation method and application thereof |
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